The cyclopropyl and oxiranyl groups are important structural elements found in a wide variety of natural products. Intrigued by the biological activities of these three-membered ring metabolites and their potential applications as biomedical agents, we have been investigating the mechanistic details of how these small ring metabolites are processed by enzymes for more than a decade. In the past few years, we have concentrated our efforts on the inactivation of acyl-CoA dehydrogenases and enoyl-CoA hydratases by cyclopropane derivatives. Both studies are particularly relevant for the development of specific inhibitors as possible therapeutic elements in the regulation of fatty acid metabolism. A PLP-dependent enzyme, 1- aminocyclopropane-1-carboxylate deaminase, which catalyzes the ring cleavage of a variety of cyclopropyl amino acids, was also studied. This work led to the discovery of a unique mechanism for coenzyme B6 catalysis. Study of cyclopropane fatty acid (CFA) synthase, started in the current grant period, represents our first foray into the biosynthesis of three membered ring compounds. This mechanistically intriguing enzyme is a potential target for developing treatment of drug-resistant bacterial infections. To explore the construction of three-membered ring systems other than carbocycles, we have recently initiated a study of oxiranyl ring formation catalyzed by (S)-2-hydroxypropylphosphonate (HPP) epoxidase in the biosynthesis of antibiotic fosfomycin. This epoxidation is unusual since it is effectively a dehydrogenation reaction of a secondary alcohol. This enzyme holds potential to be used to prepare bioactive agents containing a reactive oxirane group. Outlined in this proposal are our future plans to further characterize the reactions catalyzed by CFA synthase and HPP epoxidase. The designed experiments cover the elucidation of the mechanism of CFA synthase and the biochemical details of its interracial catalysis, the study of the biochemical properties of HPP epoxidase, including the chemical nature of the iron-catecholate core as well as the putative protein radical, and the mechanism of its catalyzed oxirane ring closure step. Since these two enzymes are each prototypes of an important class of catalysts, many of which are biomedically relevant, the proposed research will not only aid in delineating the mechanisms of the target enzymes, but may also provide valuable information for designing strategies to control and mimic the catalytic roles of other related enzymes. Thus, our results are expected to contribute to the broad field of bioorganic chemistry and also hold potential for future clinical applications.